Project Summary Tuberculosis (TB), an infectious disease caused by species of Mycobacterium tuberculosis complex (MTC), remains one of the major public health problems worldwide, with ~10 million new cases and about ~1.5 million deaths each year. With ~70,000 new cases reported in 2017, Brazil remains among the 22 high-burden countries that account for >80% of all TB cases worldwide. Non-tuberculous mycobacteria (NTM) are an emerging group of related opportunistic pathogens that cause TB-like pulmonary infections, particularly in patients with underlying comorbidities such as HIV or cystic fibrosis. NTM infections are notoriously refractory to treatment, with <50% cure rates for some species despite year-long treatment regimens with antibiotic cocktails of oral and injectable drugs. This situation is further exacerbated by HIV co-infection which promotes disease progression and complicates diagnosis of TB and NTM pulmonary infections. An estimated 3.5 million cases of TB go undetected each year, largely due to the lack of affordable, effective point-of-care diagnostics for TB/NTM infection. As a result, infected patients develop more severe disease and continue to transmit these pathogens. The frequent misdiagnosis of NTM infections as TB by the commonly used smear microscopy method delays appropriate treatment which can result in worse clinical outcomes. Thus, point-of-care (POC) tests are urgently needed to provide physicians with actionable data required to effectively treat patients with these debilitating chronic diseases. The goal of this project is to take advantage of recent advancements in DNA nanotechnology, molecular sensors, and 3D-printing to significantly improve POC diagnostics capability for TB and NTM. Our platform combines the speed and sensitivity of RPA isothermal amplification, the specificity of binary deoxyribozyme (BiDz) sensors, and economy and portability of a 3D-printed assay device. We will optimize multi-color multiplex sensor assays for specific detection of TB and NTM pathogens from sputum samples. A cheap, portable battery-powered device for assay incubation and fluorescent detection built using 3D-printing technology will be tested. Finally, ?real-world? validation of this diagnostic assay will be conducted in Brazil on samples from HIV+ and HIV- cohorts. If successful, this flexible technology could be exploited to devise POC diagnostic assay for other infectious diseases. An added outcome of this international collaboration will be enhancement of the infectious disease research capacity and infrastructure in Brazil.